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Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease.

Lee DI, Zhu G, Sasaki T, Cho GS, Hamdani N, Holewinski R, Jo SH, Danner T, Zhang M, Rainer PP, Bedja D, Kirk JA, Ranek MJ, Dostmann WR, Kwon C, Margulies KB, Van Eyk JE, Paulus WJ, Takimoto E, Kass DA - Nature (2015)

Bottom Line: PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS.Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation.Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA.

ABSTRACT
Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

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Development of PDE9A Knockout (PDE9A−/−), and specificity of PDE5A or PDE9A siRNAa, PDE9A knockout (PDE9A−/−) mice were developed by replacing of Exon-12 region with Lac-Z-neomycin in the catalytic domain of c-terminal in PDE9a gene. The genotyping was performed using specific primers designed between Exon-11 and -13 including neomycin as following; GS1 (5′-cacagatgatgtacagtatggtctgg-3′), GS2 (5′-tgcagtcatcaggaccaagatgtcc-3′) and Neo (5′-gacgagttcttctgaggggatcgatc-3′). b, The typical genotyping pattern of PDE9A−/− mice was shown on 2% agarose gel (250 bp for WT and 500 bp for PDE9A−/− mice). c, Selective gene silencing using siRNA targeting PDE5A or PDE9A. PCR confirms specificity and substantial gene knockdown achieved in cell culture (n=6/group).
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Figure 5: Development of PDE9A Knockout (PDE9A−/−), and specificity of PDE5A or PDE9A siRNAa, PDE9A knockout (PDE9A−/−) mice were developed by replacing of Exon-12 region with Lac-Z-neomycin in the catalytic domain of c-terminal in PDE9a gene. The genotyping was performed using specific primers designed between Exon-11 and -13 including neomycin as following; GS1 (5′-cacagatgatgtacagtatggtctgg-3′), GS2 (5′-tgcagtcatcaggaccaagatgtcc-3′) and Neo (5′-gacgagttcttctgaggggatcgatc-3′). b, The typical genotyping pattern of PDE9A−/− mice was shown on 2% agarose gel (250 bp for WT and 500 bp for PDE9A−/− mice). c, Selective gene silencing using siRNA targeting PDE5A or PDE9A. PCR confirms specificity and substantial gene knockdown achieved in cell culture (n=6/group).

Mentions: To test PDE9A involvement in the heart, gene and protein expression were assessed in myocardial tissue and isolated myocytes. Fig. 1a shows PDE9A immunostaining in rat neonatal cardiomyocytes (RNCMs) and adult mouse myocytes, with targeted gene deletion (siRNA or PDE9A−/− mice, Extended Data Fig. 1) as a negative control. Protein detection by immunoblot in neonatal myocytes is shown in Extended Data Fig. 2a. Basal gene expression is low but increases with agonist (e.g. phenylephrine (PE)) or mechanical (in vivo pressure-overload) stimulation (Fig. 1b). Increased PDE9A protein expression and cGMP-esterase activity is found in left ventricular (LV) myocardium from humans with heart failure and depressed function (Fig. 1c–e, Extended Data Fig. 2b, c and Extended Data Table 1). Protein expression also increases in human LV hypertrophy from aortic stenosis (pressure-overload), and most strikingly in heart failure and a preserved ejection fraction (HFPEF, Fig. 1f), a prevalent form of HF wherein contractile function appears normal despite symptoms12. Human PDE9A expression primarily localizes to myocytes based on co-localization with troponin-T (Fig. 1g–i) and in situ hybridization staining (Fig. 1j, k). Whereas Pde5a is expressed in fibroblasts9, Pde9a is essentially undetectable in isolated human fibroblasts (qPCR-threshold cycle= 39).


Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease.

Lee DI, Zhu G, Sasaki T, Cho GS, Hamdani N, Holewinski R, Jo SH, Danner T, Zhang M, Rainer PP, Bedja D, Kirk JA, Ranek MJ, Dostmann WR, Kwon C, Margulies KB, Van Eyk JE, Paulus WJ, Takimoto E, Kass DA - Nature (2015)

Development of PDE9A Knockout (PDE9A−/−), and specificity of PDE5A or PDE9A siRNAa, PDE9A knockout (PDE9A−/−) mice were developed by replacing of Exon-12 region with Lac-Z-neomycin in the catalytic domain of c-terminal in PDE9a gene. The genotyping was performed using specific primers designed between Exon-11 and -13 including neomycin as following; GS1 (5′-cacagatgatgtacagtatggtctgg-3′), GS2 (5′-tgcagtcatcaggaccaagatgtcc-3′) and Neo (5′-gacgagttcttctgaggggatcgatc-3′). b, The typical genotyping pattern of PDE9A−/− mice was shown on 2% agarose gel (250 bp for WT and 500 bp for PDE9A−/− mice). c, Selective gene silencing using siRNA targeting PDE5A or PDE9A. PCR confirms specificity and substantial gene knockdown achieved in cell culture (n=6/group).
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Related In: Results  -  Collection

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Figure 5: Development of PDE9A Knockout (PDE9A−/−), and specificity of PDE5A or PDE9A siRNAa, PDE9A knockout (PDE9A−/−) mice were developed by replacing of Exon-12 region with Lac-Z-neomycin in the catalytic domain of c-terminal in PDE9a gene. The genotyping was performed using specific primers designed between Exon-11 and -13 including neomycin as following; GS1 (5′-cacagatgatgtacagtatggtctgg-3′), GS2 (5′-tgcagtcatcaggaccaagatgtcc-3′) and Neo (5′-gacgagttcttctgaggggatcgatc-3′). b, The typical genotyping pattern of PDE9A−/− mice was shown on 2% agarose gel (250 bp for WT and 500 bp for PDE9A−/− mice). c, Selective gene silencing using siRNA targeting PDE5A or PDE9A. PCR confirms specificity and substantial gene knockdown achieved in cell culture (n=6/group).
Mentions: To test PDE9A involvement in the heart, gene and protein expression were assessed in myocardial tissue and isolated myocytes. Fig. 1a shows PDE9A immunostaining in rat neonatal cardiomyocytes (RNCMs) and adult mouse myocytes, with targeted gene deletion (siRNA or PDE9A−/− mice, Extended Data Fig. 1) as a negative control. Protein detection by immunoblot in neonatal myocytes is shown in Extended Data Fig. 2a. Basal gene expression is low but increases with agonist (e.g. phenylephrine (PE)) or mechanical (in vivo pressure-overload) stimulation (Fig. 1b). Increased PDE9A protein expression and cGMP-esterase activity is found in left ventricular (LV) myocardium from humans with heart failure and depressed function (Fig. 1c–e, Extended Data Fig. 2b, c and Extended Data Table 1). Protein expression also increases in human LV hypertrophy from aortic stenosis (pressure-overload), and most strikingly in heart failure and a preserved ejection fraction (HFPEF, Fig. 1f), a prevalent form of HF wherein contractile function appears normal despite symptoms12. Human PDE9A expression primarily localizes to myocytes based on co-localization with troponin-T (Fig. 1g–i) and in situ hybridization staining (Fig. 1j, k). Whereas Pde5a is expressed in fibroblasts9, Pde9a is essentially undetectable in isolated human fibroblasts (qPCR-threshold cycle= 39).

Bottom Line: PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS.Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation.Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA.

ABSTRACT
Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Show MeSH
Related in: MedlinePlus